The microbial community in the human large intestine consists of a diverse range of bacteria that are predominantly obligate anaerobes. These bacteria act together to degrade dietary substrates that reach the colon (including insulin, fructo-oligosaccharides and resistant starch), producing a range of products that are important for human health and disease.
The mucosal immune response can be influenced by manipulation of the normal resident bacterial flora. This flora possesses a large variety of biological and immunomodulatory properties that can, directly or indirectly, influence the development and function of the mucosal immune system. Chronic disorders of the gut, for example inflammatory bowel disease (IBD) which includes the disorders Crohn's disease and ulcerative colitis, affect a significant proportion of the population in developed countries. Animal models of mucosal inflammation have been used to try and determine the immune mechanisms involved in the pathogenesis of these diseases. Chronic colitis develops spontaneously in interleukin (IL) 2−/− and IL10−/− mice both of which are used as models of IBD. Many other mouse models of IBD have also been described, most of which have targeted deletions of immune response genes. Current treatment of IBD is restricted to anti-inflammatory and immunosuppressive drug therapies including recombinant IL10 and antibodies to tumour necrosis factor-α (TNF-α). However, these therapies are not curative and may cause adverse side effects such as toxicity and immunosuppression. Therefore, there is a need for a more targeted and controlled form of immunotherapy.
It is known from the prior art to use commensal, or bacteria that occur naturally in the alimentary canal, such as Lactobacillus spp. and Streptococcus spp. to treat intestinal inflammation and certain forms of IBD in humans (Shanahan 2001), however these results have limited evidence of success and inconsistent efficacy. It is also known from the prior art to use genetically engineered food grade Lactococcus lactis to secrete interleukin-10 (IL10), which when administered intragastrically to two murine models of IBD was shown to be as effective in both preventing and treating disease as the more conventional steroid therapy (Steidler et al. 2000). This Lactococcus system has also been used to produce biologically active IL2 and IL6 (Steidler et al. 1995; Steidler et al. 1998). However, a major disadvantage associated with these prior art systems is that L. lactis is not able to colonise the gut due to the inability of the organism to bind to the gut epithelium and/or its nutritional dependence on the provision of amino acids and peptides which are unavailable in vivo. Accordingly any in vivo treatment or therapy would require repeated dosing to the appropriate site with the modified organism.
Another biosafety concern and disadvantage of the use of this particular aerobic bacterium is that it could survive outside of the host/patient for sufficient time to be transmitted to others.
A yet further disadvantage of the prior art systems is that there is no means of controlling the constitutive expression of the immunologically active interleukin molecules and these active molecules themselves when overproduced, can have adverse effects. Accordingly the prior art genetically modified probiotic systems lack control and regulation of the activity of probiotic bacteria after administration. This represents a serious safety issue for human therapy.
To address the deficiencies in the prior art and to further develop commensal bacteria as novel delivery systems for biologically active molecules, we have developed genetically engineered probiotic organisms in which the production of immunotherapeutic agents by commensal bacteria in situ can be regulated and controlled by dietary factors.
It is an object of the present invention to engineer a gut commensal bacterium so as to produce and secrete biologically active polypeptide(s) or protein(s) in a regulated manner as a basis for novel immunotherapies for chronic gut disorders.